SHBG141–161 Domain-Peptide Stimulates GPRC6A-Mediated Response in Leydig and β-Langerhans cell lines

Abstract

AbstractGPRC6A is acknowledged as a major regulator of energy metabolism and male fertility through the action of undercarboxylated osteocalcin (ucOCN), representing a possible therapeutic target. We recently showed that the sex hormone-binding globulin (SHBG) binds to GPRC6A through the likely involvement of the 141–161 domain. To confirm this model, here we investigated the possible binding and agonist activity of SHBG(141–161) domain-peptide (SHBG141–161) on GPRC6A. The binding of SHBG141–161 to GPRC6A and downstream dissociation from Gαi(GDP) protein was computationally modelled. SHBG141–161 was obtained by solid-phase synthesis, characterized by circular dichroism (CD) and the receptor binding was assessed by displacement of ucOCN on HEK-293 cells transfected with GPRC6A gene. Agonist activity of SHBG141–161 was assessed on Leydig MA-10 and Langerhans β-TC6 cell lines through the GPRC6A-mediated release of testosterone (T) and insulin. SHBG141–161 was predicted to bind to GPRC6A and to reduce the affinity for Gαi(GDP) at computational level. Conformational properties and binding to GPRC6A of the synthetic SHBG141–161 were confirmed by CD and displacement experiments. SHBG141–161 stimulated cell secretion of T and insulin, with dose dependency from 10−13 to 10−11M for T release (respectively P = 0,041 10−13M; P = 0,032 10−12M; P = 0,008 10−11M vs basal) and for 10−12 to 10−10M for insulin (respectively P = 0,041 10−12M; P = 0,007 10−11M; P = 0,047 10−10M; P = 0,045 vs basal). Blockade with anti GPRC6A IgG abolished the response to SHBG141-161, suggesting agonist specificity. SHBG141–161 showed stimulating activity on GPRC6A, representing a template peptide with possible therapeutic use for metabolic and endocrine disorders.